Effects of α2-adrenoceptor stimulation on luminal alkalinisation and net fluid flux in rat duodenum

The sympathetic nervous system is highly involved in the regulation of gastrointestinal functions such as luminal alkalinisation and fluid absorption. However, the exact mechanisms are not clear. This study aimed to delineate how α2-adrenergic receptor stimulation reduces duodenal luminal alkalinisation and induces net fluid absorption. This was tested by perfusing the duodenum of anesthetized rats with isotonic solutions devoid of Cl- and/or Na+, in the absence and presence of the α2-adrenoceptor agonist clonidine. The clonidine was also studied in rats treated with dimethylamiloride (a Na+/H+ exchange inhibitor), vasoactive intestinal peptide, and the nicotinic receptor antagonist hexamethonium. Clonidine reduced luminal alkalinisation and induced net fluid absorption. The Cl--free solution decreased luminal alkalinisation and abolished net fluid absorption, but did not prevent clonidine from doing so. Both the Na+-free solution and luminal dimethylamiloride increased luminal alkalinisation and abolished net fluid absorption, effects counteracted by clonidine. The NaCl-free solution (D-mannitol) did not affect luminal alkalinisation, but reduced net fluid absorption. Clonidine reduced luminal alkalinisation and induced net fluid absorption in rats perfused luminally with mannitol. However, clonidine did not affect the vasoactive intestinal peptide-induced increase in luminal alkalinisation or fluid secretion. Pre-treatment with hexamethonium abolished the effects of clonidine on luminal alkalinisation and net fluid flux. In summary, our in vivo experiments showed that clonidine-induced reduction in luminal alkalinisation and induction of net fluid absorption was unrelated to luminal Na+ and Cl-, or to apical Na+/H+ or Cl-/HCO3- exchangers. Instead, clonidine seems to exert its effects via suppression of nicotinic receptor-activated acetylcholine secretomotor neurons.


Introduction
Sympathetic postganglionic nerve fibers enter the intestinal wall along arteries. These fibers terminate primarily in the myenteric and submucosal plexuses, but some also penetrate into the submucosa and mucosa [1]. Upon activation of these neurons, noradrenalin is released, which binds to two main types of adrenergic receptors, α and β. This activation inhibits peristalsis, reduces blood flow, and increases fluid absorption. maintained under constant conditions (12:12 h light-dark cycles; 21˚C) with ad libitum access to food and water. Before the experiments, the rats were fasted (in pairs) overnight with free access to water. Thereafter they were anaesthetized with an intraperitoneal injection of 125 mg kg -1 thiobutabarbital sodium salt (Inactin, St. Louis, MO, USA). Body temperature was maintained at 37.5 ± 0.5˚C during the surgical procedure. The single-pass duodenal perfusion experiment was the same as described in [29]. At the end of the perfusion experiment, rats were sacrificed with a i.v. injection with saturated KCl solution.

Measurement of duodenal luminal alkalinisation
The luminal alkalinisation was assessed by back titration as described previously [30], and expressed as micromoles of base transported per cm 2 serosal surface area per hour (μmol cm -2 h -1 ).

Measurement of fluid flux
The method to assess transepithelial net fluid flux is detailed in [30]. In brief, the absolute flux was determined by subtracting the collected effluent volume from the peristaltic pump volume. The net change in fluid flux in response to the test solution was determined as follows. The mean of the effluent volumes sampled before the exposure to the test solution was subtracted from the mean value in response to the test solution, in relationship to the weight of the duodenum as determined after the experiment. Fluid flux was expressed in ml per g wet tissue weight per hour (ml g -1 h -1 ). The drift of the peristaltic pump over time was insignificant (<0.1%).

Experimental protocol
The single-pass intestinal perfusion setups with treatments and luminal conditions are shown in Fig 1. Mean arterial blood pressure (MABP), the rate of luminal alkalinisation and the transepithelial net fluid flux were all assessed.

Clonidine with and without idazoxan
The duodenum was perfused luminally with a 155 mM NaCl solution throughout the 90-min experiment. Thirty min after the start of the experiment, clonidine was administered intravenously as a continuous infusion (1.0 ml h -1 ) at a dose of 10 or 50 μg kg -1 h -1 . A third subgroup used the same protocol as the clonidine 10 μg kg -1 h -1 except that idazoxan, an α 2adrenoceptor antagonist, was administered intravenously (100 μg kg -1 bolus + 100 μg kg -1 h -1 infusion) starting 30-min before the start of the experiment.

Perfusion with a Cl --free Na 2 SO 4 solution
After the initial 30-minute isotonic NaCl perfusion, the duodenum was perfused with a Cl -free isotonic Na 2 SO 4 solution (75 mM Na 2 SO 4 with 60 mM D-mannitol) for 100 min. The protocol was the same for the second subgroup except that clonidine was given by an intravenous infusion (1 ml h -1 ) at a dose of 10 μg kg -1 h -1 , starting 40 min after commencement of the Na 2 SO 4 perfusion and continued for 60 min.

Perfusion with a Na + -free NMDG solution
The experimental protocol was exactly the same as for the Cl --free perfusion (Na 2 SO 4 ), except that the solution was a Na + -free isotonic N-methyl-D-glucamine chloride (NMDG-Cl; 155 mM; 285-291 mOsm kg -1 H 2 O).

Perfusion with a Na + /H + exchange inhibitor, with and without luminal Cl -
After the 30-min perfusion with isotonic NaCl, the duodenum was perfused for 100 min with the Na + /H + exchange inhibitor DMA (1 mM) in either an isotonic NaCl solution or the Cl -free isotonic solution described above. The protocol for the third subgroup was the same except that clonidine was administered intravenously starting 40 min after commencement of the perfusion with the DMA or the Cl --free DMA solution and continued for 60 min.

Perfusion with a NaCl-free D-mannitol solution
After an initial 30-min period of isotonic NaCl perfusion, the duodenum was perfused with an isotonic D-mannitol solution (260 mM D-mannitol; 286-290 mOsm kg -1 H 2 O). In the second subgroup, clonidine was administered intravenously at a dose of 10 μg kg -1 h -1 starting 40 min after commencement of the mannitol perfusion and continued for 60 min.

Effects of vasoactive intestinal peptide
Animals were divided in two subgroups. In both groups, the duodenum was perfused with isotonic NaCl throughout the experiment. In the first subgroup, vasoactive intestinal peptide (VIP) was intravenously infused at a rate of 15 μg kg -1 h -1 starting 30 min after commencement of effluent collection and continued for 120 min. Sixty min after the start of the VIP infusion, clonidine was continuously infused at 10 μg kg -1 h -1 throughout the experiment. In the second subgroup, the clonidine was administered 30 min after start of effluent collection. Sixty min after commencement of the clonidine infusion, VIP was administered intravenously at 15 μg kg -1 h -1 throughout the experiment.

Effects of hexamethonium
The duodenum was perfused with isotonic NaCl throughout the experiment. Thirty min after initiation of the perfusion, hexamethonium, a non-selective competitive nicotinic receptor antagonist, was administered intravenously as a bolus at 10 mg kg -1 followed by a continuous infusion at 10 mg kg -1 h -1 . In the second subgroup, clonidine was given by an intravenous infusion (1 ml h -1 ) at a dose of 10 μg kg -1 h -1 , starting 40 min after start of the hexamethonium infusion and continued for 60 min.

Statistical analyses
Values are expressed as means ± SEM. The statistical significance of the data was tested by analysis of variance (ANOVA) followed by Tukey's Multiple Comparison test. To test differences within a group, i.e. comparing the results obtained before, during, and after perfusion with the different solutions, a one-factor repeated measures ANOVA was used. Differences between two groups of animals was tested by students t-test, and when multiple comparisons were needed an unpaired two-factor repeated measures ANOVA was used. All statistical analyses were performed using GraphPad Prism software. P<0.05 was considered as significant (two-tailed test). The data that support the findings of this study are available from the corresponding author upon reasonable request.

Basal luminal alkalinisation and fluid flux
The duodenum in all groups was perfused with isotonic NaCl for 30 min. The mean basal rate of luminal alkalinisation was 7.1 ± 2.7 μmol cm -2 h -1 and the basal net fluid flux was -0.81 ± 1.18 ml g -1 h -1 (mean ± SD, n = 105 for both). The net fluid flux was significantly below zero (i.e. net fluid absorption, P<0.001), which is depicted with a minus sign in text and figures. There was no linear correlation (r 2 = 0.02, P = 0.13) between the basal absolute rate of luminal alkalinisation and the basal net fluid flux.

Effect of α2-adrenoceptor stimulation in the absence of luminal Cl -
The aim was to determine whether the effects of clonidine on luminal alkalinisation and net fluid flux are dependent on apical chloride HCO 3 exchange. To examine this, the duodenum was perfused with an isotonic Cl --free Na 2 SO 4 solution in the absence and presence of clonidine. The isotonic Cl --free Na 2 SO 4 solution decreased (P<0.01) luminal alkalinisation and changed (P<0.001) net fluid flux from net absorption to values not different from zero (Fig 3A  and 3B). Interestingly, the magnitude of the decrease in luminal alkalinisation in response to the Cl --free Na 2 SO 4 solution varied greatly between the animals and was linearly correlated to basal luminal alkalinisation (y = 1.68-0.64x, r 2 = 0.70, P<0.001 and n = 14) ( Fig 4A).
In animals perfused with the Cl --free Na 2 SO 4 solution clonidine decreased (P<0.01) luminal alkalinisation further (by 52%) and induced (P<0.001) net fluid absorption (Fig 3C and  3D). The changes in luminal alkalinisation and net fluid flux were both significantly greater in the animals treated with Na 2 SO 4 and clonidine than with the Na 2 SO 4 alone (Fig 3E and 3F).

Effect of α 2 -adrenoceptor stimulation in the absence of luminal Na +
The objective was to determine whether the effects of clonidine on duodenal luminal alkalinisation and net fluid flux are dependent on apical Na + -H + exchange. This was done by perfusion of the duodenum with an isotonic Na + -free NMDG chloride solution in the absence and presence of clonidine. This solution increased (P<0.05) luminal alkalinisation and changed (P<0.05) basal net fluid flux from net absorption to zero (Fig 5A and 5B). No linear correlation was found between the basal luminal alkalinisation and the NMDG-induced increase in luminal alkalinisation (r 2 = 0.19, P = 0.22, n = 10) ( Fig 4B).
In animals perfused with an isotonic Na + -free NMDG chloride solution clonidine decreased (P<0.001) luminal alkalinisation and changed (P<0.01) net fluid flux from a value not different from zero to net fluid absorption (Fig 5C and 5D). The changes in luminal alkalinisation and the net fluid flux were both significantly greater in NMDG plus clonidine treated animals than in those treated with NMDG alone (Fig 5E and 5F).

Effect of α 2 -adrenoceptor stimulation in the presence of luminal dimethylamiloride
The aim was to determine whether the effects of clonidine on luminal alkalinisation and net fluid flux are affected by luminal dimethylamiloride (DMA), a non-specific inhibitor of Na + / H + exchange. DMA increased luminal alkalinisation (P<0.05), and changed (P<0.01) net fluid flux from a basal value not different from zero towards net fluid secretion (Fig 6A and  6B). No linear correlation was found between the basal luminal alkalinisation and the DMAinduced increase in luminal alkalinisation (r 2 = 0.12, P = 0.25, n = 13) ( Fig 4C).
In animals perfused with DMA clonidine significantly decreased the rate of luminal alkalinisation and induced net fluid absorption (Fig 6C and 6D). The changes in luminal alkalinisation and net fluid flux were both significantly greater in DMA plus clonidine treated rats than in those treated with DMA alone (Fig 6E and 6F). The effect of α2-adrenoceptor stimulation in the absence of luminal NaCl The objective was to determine whether the effects of clonidine on luminal alkalinisation and net fluid flux is affected by the lack of luminal NaCl. To achieve this, the duodenum was perfused with an isotonic NaCl-free solution, i.e., an isotonic D-mannitol solution. The isotonic D-mannitol solution had no significant effect on the mean luminal alkalinisation (Fig 7a). However, a very good (P<0.001) linear correlation (y = 2.08-0.52x, r 2 = 0.86) was found between the basal luminal alkalinisation and the mannitol-induced change in luminal alkalinisation ( Fig 4D). During the perfusion with isotonic NaCl there was a net fluid absorption, which decreased significantly in response to the isotonic mannitol solution (Fig 7B). The In animals perfused with the isotonic NaCl-free solution clonidine significantly decreased luminal alkalinisation and increased net fluid absorption (Fig 7C and 7D). The changes in luminal alkalinisation and the net fluid flux were both significantly greater in mannitol plus clonidine treated animals than in mannitol treated ones (Fig 7E and 7F).
The effects of α 2 -adrenoceptor stimulation in the absence of luminal Cland in the presence of dimethylamiloride. To further examine whether the effects induced by clonidine Perfusion with isotonic Cl --free Na 2 SO 4 plus DMA did not affect the mean luminal alkalinisation (Fig 8A). However, similar to the mannitol-perfusion experiments, a very good (P<0.001) linear correlation (y = 3.31-0.62x, r 2 = 0.79, n = 11) was found between the basal luminal alkalinisation and the Na 2 SO 4 plus DMA-induced change in luminal alkalinisation (Fig 4E). The Na 2 SO 4 plus DMA solution changed (P<0.05) net fluid flux from a value not different from zero to net fluid secretion (Fig 8B). In animals perfused with the Cl --free Na 2 SO 4 plus DMA solution clonidine reduced luminal alkalinisation and abolished the Na 2 SO 4 plus DMA-induced net fluid secretion (Fig 8C  and 8D). The changes in luminal alkalinisation and the net fluid flux were both significantly greater in clonidine treated animals (Fig 8E and 8F).   (Fig 9A and 9B). In VIP-treated animals clonidine decreased, but did not abolish, luminal alkalinisation (P<0.05) and reduced net fluid secretion (P<0.01).
In a second series of experiments we examined whether VIP could increase electrolyte fluid secretion in the presence of clonidine. Indeed, VIP increased (P<0.001) luminal alkalinisation and changed (P<0.001) net fluid flux from net fluid absorption to net secretion (Fig 9C and  9D). The net increase in luminal alkalinisation and the change in net fluid flux in response to VIP were virtually the same in animals treated with VIP alone as in those pre-treated with clonidine (Fig 9E and 9F). The net decrease in luminal alkalinisation and the change in net fluid flux in response to clonidine were virtually the same in animals treated with clonidine alone and in those pre-treated with VIP (Fig 9G and 9H).

Does α 2 -adrenoceptor stimulation affect the nAChR-induced inhibition on fluid flux and luminal alkalinisation?
Clonidine may indirectly inhibit electrolyte fluid secretion by suppressing nicotinic acetylcholine receptors-activated (nAChR) secretomotor neurons that innervate the epithelium. If this were the case, treatment with hexamethonium would prevent clonidine from inhibiting luminal alkalinisation and inducing net fluid absorption. Administration of hexamethonium promptly and continuously reduced MABP (Fig 10A). Concomitantly, hexamethonium decreased luminal alkalinisation and augmented net fluid absorption (Fig 10B and 10C). A very good correlation was found between the basal luminal alkalinisation and the hexamethonium-induced decrease in luminal alkalinisation (y = 2.04-0.69x, r 2 = 0.77, P<0.001, n = 15), (Fig 10D). Intravenous infusion of clonidine to rats pre-treated with hexamethonium had no effect on MABP (Fig 10A), luminal alkalinisation, or net fluid flux (Fig 10E and 10F).

Summary of treatment effects on fluid flux and luminal alkalinisation
Interventions and their effects on fluid absorption and luminal alkalinisation in the villi and crypt regions are summarized in Fig 11.

Discussion
The aim of the present investigation was to shed further light on the mechanism by which α 2adrenoceptor stimulation inhibits luminal alkalinisation and induces net fluid absorption in the rat duodenum in vivo. More specifically, we wanted to answer if (i) the effects of clonidine on electrolyte and water transport were due to increased absorption or reduced secretion or a combination of both, (ii) if the effects of clonidine were sensitive to the removal of luminal Cland/or Na + , and (iii) if clonidine exerted its effects via suppression of excitatory nicotinergic receptor-activated secretomotor neurons [3].
Clonidine at two doses induced the same inhibition of luminal alkalinisation and induction of net fluid absorption. The fact that the effects were markedly attenuated by the α2-adrenoceptor antagonist idazoxan, strongly suggests that clonidine exerts its effects via α2-adrenoceptors. An interesting observation was that clonidine was less effective in reducing luminal alkalinisation and augmenting net fluid absorption in rats with a low basal rate of alkalinisation or a high basal net fluid absorption, respectively, which most likely reflects a higher basal sympathetic tone to the duodenal segment in these rats.
Previous in vivo experiments in rodent duodenum have shown that ablation of Cl -/HCO 3 exchangers (Slc26a6 or Slc26a3) reduces basal duodenal mucosal HCO 3 secretion, and that luminal perfusion with a Cl --free solution markedly reduces luminal alkalinisation [18,30]. This suggests that Cl -/HCO 3 exchangers play an important role in regulating duodenal mucosal HCO 3 secretion. The results in our study clearly showed that the magnitude of the Cl --free  [31] found that the PGE 2 -and the carbachol-stimulated duodenal HCO 3 secretion was reduced in Slc26A6-deficient mice in vitro. Other experiments in vivo in the rat duodenum show that the motility-induced stimulation of duodenal luminal alkalinisation is almost completely abolished by removal of luminal Cl - [30]. The Cl --free solution-induced decrease in luminal alkalinisation was associated with abolishment of net fluid absorption. This confirms previous findings in humans and rodents that luminal Cland the presence of functional Cl -/HCO 3 exchangers are required for a normal intestinal fluid absorption [32][33][34]. In the present study it is clearly shown that clonidine significantly reduced luminal alkalinisation and induced net fluid absorption also rats perfused with a Cl --free solution, strongly suggesting a mechanism independent of luminal Cl -.
Luminal perfusion of the duodenal segment with the Na + -free solution, or inhibition of Na + /H + exchange (DMA), increased luminal alkalinisation, confirming findings from rat and mouse [17,18]. This probably occurs via inhibition of apical H + efflux, which then unmasks the activity of Cl -/HCO 3 exchangers and the CFTR channel. In contrast to the Cl --free solution, no linear correlation was found between the basal rate of luminal alkalinisation and the Na + -free or DMA-induced increase in luminal alkalinisation. One explanation may be a predominance of Cl -/HCO 3 exchangers over Na + /H + exchangers in duodenum [35,36]. Another explanation may be that the activity of apical Na + /H + exchangers is less variable than that of the Cl -/HCO 3 exchangers in our in vivo duodenal-perfusion model.
Luminal perfusion with the Na + -free (NMDG) or the DMA solution abolished net fluid absorption, in agreement with findings that the lack of luminal Na + , NHE3-gene knockout [34], or NHE3 inhibition by S1611 [18], reduce fluid absorption in the mouse small intestine. Clonidine may induce net fluid absorption and inhibit luminal alkalinisation by stimulation of basal NHE3, and/or by inhibiting the cAMP-and Ca 2+ -induced decreases in apical NHE3 activity [37]. Our data showed that clonidine effectively reduced luminal alkalinisation and induced net fluid absorption also in the absence of luminal Na + and in the presence of DMA, suggesting a mechanism independent of Na + /H + exchange.
The fact that the Cl --free (Na 2 SO 4 ) solution decreased luminal alkalinisation, while the Na + -free one (NMDG) increased it made us curious what would happen in response to luminal perfusion with the NaCl-free mannitol solution, or the Cl --free plus DMA solution. It turned out that these two solutions had no significant effect on luminal alkalinisation. However, the change in luminal alkalinisation of the two treatments depended on the rate of basal luminal alkalinisation. In rats with a spontaneously low basal alkalinisation, the decrease in luminal alkalinisation induced by the removal of Clwas probably counterbalanced by the increase in alkalinisation brought about by the lack of luminal Na + , or by the inhibition of Na + /H + exchange. Clonidine also decreased luminal alkalinisation in the absence of luminal NaCl, and luminal Clplus inhibition of Na + /H + exchange. The mannitol solution markedly attenuated the net fluid absorption while the Cl --free plus DMA solution induced net fluid secretion. Clonidine augmented the net fluid absorption in mannitol-perfused animals and abolished the net fluid secretion in those perfused with Na 2 SO 4 plus DMA. Taken together, these results further suggested that clonidine reduces luminal alkalinisation and induces net fluid absorption by a mechanism independent of Cl -/HCO 3 and/ Na + /H + exchange.
How then does clonidine reduce luminal alkalinisation and induce net fluid absorption? The most obvious explanation is that clonidine inhibits electrolyte fluid secretion. Most likely this occurs by inhibition of CFTR or other Clchannels in the crypt region of the epithelium, where α2-adrenoceptors are predominately expressed, both in rat jejunum and human duodenum [38,39]. We tested this hypothesis by examining the ability of clonidine to inhibit the effects of VIP, which is normally found in nerve fibres in proximity to the duodenal epithelium [40,41]. It has previously been shown that VIP stimulates duodenal mucosal HCO 3 transport [42][43][44], as well as fluid secretion in animals with functional CFTR activity [27,45]. In guinea pig jejunum in vitro, VIP appears to exert its stimulatory effect on secretion (short-circuit current), predominately via activation of VPAC1 receptors located in the mucosa and partly via an action on submucosal neurons [46]. If VIP and clonidine exert their actions on the same target cell, it seems reasonable to assume that clonidine would reduce the stimulatory effect of VIP, and that VIP would reduce the inhibitory action of clonidine. In the first series of experiments, clonidine reduced both the VIP-induced increase in luminal alkalinisation and the increase in net fluid secretion, in agreement with data from rat jejunum in vivo [47]. However, in animals pre-treated with clonidine, the VIP-induced increase in luminal alkalinisation and net fluid secretion were not different from what was obtained with VIP alone. Furthermore, the net decrease in luminal alkalinisation and the change in net fluid flux in response to clonidine were virtually the same in animals treated with clonidine alone as in those pre-treated with VIP. It thus appears that clonidine does not affect the VIP-induced stimulation of secretion but rather the basal secretion, which may be regulated by a different neural mechanism, at least in rat duodenum.
The modest, if any, inhibitory effect of clonidine on the VIP-induced stimulation of electrolyte fluid secretion raised the possibility that clonidine inhibits electrolyte fluid secretion indirectly, possibly via activation of α 2 -adrenoceptors on cholinergic enteric secretomotor neurons. We know from previous experiments that the non-selective nicotinic receptor antagonist, hexamethonium, reduces basal luminal alkalinisation [25,26], which was confirmed in the present study. The degree of inhibition of luminal alkalinisation by hexamethonium highly correlated to basal rates of luminal alkalinisation, which possibly reflects variation in secretomotor neuron activity to the epithelium. Furthermore, here we showed that hexamethonium augmented net fluid absorption by inhibiting secretion. If clonidine exerts its action solely by blocking the activity in these hexamethonium-sensitive nerves, it is reasonable to assume that it would have no effect in hexamethonium-treated rats. This turned out to be the case, which favours the notion that clonidine inhibits electrolyte-fluid secretion via suppression of the activity in excitatory secretomotor neurons, in line with findings in rat jejunum in vivo [48]. Furthermore, the data does not support a direct effect of clonidine on α2-adrenoceptors expressed on epithelial cells, as suggested from stripped ileal mucosa with the voltage clamp technique [49]. Most likely clonidine acts by reducing the release of acetylcholine by secretomotor neurons, which binds to muscarinic M3 receptors on the epithelial crypt cells (Fig 11). Acetylcholine causes intracellular Ca 2+ to increase (while VIP increases cAMP), thereby stimulating apical Clsecretion via the calcium-dependent chloride channel, as well as basolateral K + secretion [50]. These effects on ion secretion (i.e., VIP and acetylcholine) act synergistically [51,52], which may explain why clonidine was still active even at the high VIP doses used in this study. We currently have ongoing experiments including muscarinic receptor antagonist to verify this.
In conclusion, the potent α2-adrenoceptor agonist clonidine turned out to inhibit luminal alkalinisation and to induce fluid absorption or inhibit secretion, in the absence of either luminal Clor Na + or both, and in the presence of a Na + /H + exchange inhibitor. Although clonidine slightly reduced the VIP-induced stimulation of luminal alkalinisation and net fluid secretion, it did not affect the magnitude of the VIP-induced fluid secretion. The suppressive effect of clonidine on luminal alkalinisation as well as its pro-absorptive action was abolished by nicotinic receptor blockade. Collectively, these in vivo results suggest that clonidine exerts its effects predominately via inhibition of fluid secretion due to suppression of excitatory nicotinergic receptor-activated secretomotor acetylcholine neurons and probably not by direct action on epithelial cells, at least not in the rat duodenum in vivo.